Live attenuated catfish vaccine and method of making

ABSTRACT

A high throughput bioluminescence mutant screening procedure is disclosed. This procedure utilizes robotics, and bacterial luciferase to allow real-time monitoring of mutant viability. The procedure was used to decelop a live attenuated vaccine for a catfish against  E. ictaluri,  which is further claimed herein. Additionally, genes from other bacterial species are disclosed which may also be used to create vaccines.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under 2004-35204-14211 awarded by the Cooperative State Research, Education, and Extension Service, USDA. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is generally directed toward a live attenuated vaccine for catfish. More particularly, it is directed toward a method for using bioluminescence to identify genes required for host resistance in bacteria. Genes that may be used to make live attenuated vaccines in bacteria are also disclosed.

BACKGROUND OF THE INVENTION

Functional genomics has enabled high-throughput methods for identifying bacterial genes and proteins that are differentially expressed in response to host defenses. In particular, both microarrays and high throughput proteomics have been used to identify bacterial genes associated with resistance to host defenses. In addition to microarrays and proteomics, other high-throughput methods have been used to identify bacterial genes upregulated in response to phagocytosis, including differential fluorescence induction (DFI), random luciferase transcriptional fusions, and selective capture of transcribed sequences (SCOTS).

However, genes that are differentially regulated in response to a host defense are not necessarily the same as those that are required for survival. For example, not all of the genes that have increased expression following phagocytosis are required for survival in phagocytes. It is also possible that not all of the genes required for survival have enough change in expression to allow detection. Therefore, mutagenesis studies complement gene and protein expression studies and are likely to detect a unique set of genes that are required for survival.

A major hurdle in identifying bacterial mutants susceptible to host defenses is that the screening methods tend to be labor intensive. Fields et al. identified 83 S. typhimurium transposon mutants with impaired macrophage survival by screening individual transposon mutants with phagocytes in 96-well plates (Fields, P. I., Swanson, R. V., Haidaris, C. G. & Heffron, F. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci USA 83, 5189-5193 (1986)). However, this assay required bacterial quantification from each well by plate counts. Zhao et al. used the same method to identify 37 Salmonella mutants susceptible to chicken macrophages (Zhao, Y. et al. Identification of genes affecting Salmonella enterica serovar Enteritidis infection of chicken macrophages. Infect Immun 70, 5319-5321 (2002)).

Improvements to allow high-throughput mutant screening have been reported, including a method for screening of bacterial mutants using bioluminescence to identify mycobacterial genes required for survival in macrophages and a microarray-based method for screening mutants. However, none of the previously described methods allow monitoring of bacterial mutant viability at multiple time points. In addition, previous bioluminescence based methods required a bacterial lysis step and addition of extraneous luciferin substrate and ATP for determining luciferase activity, which increases handling and cost while reducing the screening efficiency.

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish, an important disease of farm-raised channel catfish. Like some other species in the Enterobacteriaceae, E. ictaluri has the ability to resist killing by professional phagocytes. In particular, E. ictaluri is resistant to channel catfish neutrophils, which is an important aspect of pathogenesis because neutrophils are the predominant cell type in channel catfish intestinal tract immune cells. The intestine is an important site of entry for E. ictaluri. E. ictaluri is also resistant to killing by the alternative complement pathway in channel catfish.

There exists a need for a high throughput method for screening bacterial mutants to be used in live attenuated vaccines, such as one against Edwardsiella ictaluri in catfish.

SUMMARY OF THE INVENTION

A high throughput bioluminescence mutant screening (BLMS) method that is not labor intensive and that allows real-time monitoring of mutant viability is disclosed. Robotics was used to array mutants into 96 well plates, reducing manual labor. In addition, bacterial luciferase was used instead of firefly luciferase, which allays the need for addition of luciferin substrate and allows real-time monitoring of mutant viability. The resulting BLMS procedure allows collection of data from multiple time points for real-time screening of bacterial mutants against multiple host defense mechanisms. We utilized BLMS to identify E. ictaluri mutants that are susceptible to killing by channel catfish neutrophils and serum. Fourteen of the mutants were attenuated in channel catfish, and those that were completely attenuated were effective as live attenuated vaccines, demonstrating the utility of BLMS for vaccine development. These vaccines are disclosed as a patentable invention. Additional genes from other bacterial species that also may be used as live attenuated vaccines are also disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages of the invention will become apparent by reference to the detailed description of preferred embodiments when considered in conjunction with the drawings, which are not to scale, wherein like reference characters designate like or similar elements to the several drawings as follows:

FIG. 1 depicts an overview of mutant screening procedures.

FIG. 2 depicts the results of vaccine efficacy trial and shows the percent mortalities resulting from vaccination. Percent mortalities are the mean of four replicate tanks per treatment. PBS is saline control, Wt is parent strain 93-146, and AQUAVAC-ESC is a commercial live attenuated vaccine. Capital letters above each bar indicate statistical groupings. Groups marked with the same capital letter do not show statistically significant differences (P<0.05).

FIG. 3 depicts the results of percent mortalities resulting from challenge with parent strain 93-146 twenty one days post-vaccination. As in the above figure, percent mortalities are the mean of four replicate tanks per treatment. PBS is saline control, Wt is parent strain 93-146, and AQUAVAC-ESC is a commercial live attenuated vaccine. Capital letters above each bar indicate statistical groupings. Groups marked with the same capital letter do not show statistically significant differences (P <0.05).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The BLMS method claimed herein was successfully used in the development of a vaccine for channel catfish against E. ictaluri. Although the foregoing embodiment describes the claimed methods as applied in vaccine development against E. ictaluri, the BLMS method is widely applicable to the screening of any bacterial species.

Technological developments in functional genomics allow detection of molecular phenotypes that evade detection at the physiological or morphological levels. We disclose a new high-throughput functional genomics tool that we call bioluminescence mutant screening (BLMS) that translates molecular genotypes (gene mutations) to physiological phenotypes (light production) in bacteria and allows application of forward genetics.

BLMS involves random transposon mutation of a bacterial strain expressing bacterial luciferase operon (luxCDABE) in a stable plasmid. Following this approach, we produced a random E. ictaluri mutant library that conditionally expresses luxCDABE genes from a stable plasmid, pAKgfplux2, which allows the tracking of mutants in a pool under different experimental conditions.

In an alternative embodiment, luxCDABE genes could also be incorporated into bacterial chromosomes for a similar BLMS purpose. Chromosomal insertion of luxCDABE operon may require more sensitive instrumentation to alleviate the reduced amount of bioluminescence produced from a single copy lux operon. Our BLMS procedure requires use of IPTG because our mutant library expresses lux operon conditionally from a lacZ promoter on pAKgfplux2, which also carries a lad^(q) suppressor gene. To eliminate use of IPTG in bacterial strains without the presence of lac^(q) gene in their genome use of a mutant library constitutively expressing lux operon from a stable plasmid, such as pAKlux2 and pAKgfplux1, would be preferred.

Through screening 2,256 mutants from E. ictaluri mutant library, we were able to identify 14 attenuated mutants at the end of in vitro BLMS and in vivo fish screening. Eight mutants were common to neutrophil and serum screening while only four and only two mutants were identified as neutrophil and serum mutants, respectively. The fourteen identified E. ictaluri mutants were first characterized in terms of their virulence and vaccine potential and later mutated genes in each mutant were determined by transposon insertion sites identification. Finally, selected mutants were compared to a commercial vaccine (AQUAVAC-ESC) to determine their commercial value. Findings from our research suggest that BLMS is a very powerful screening method for development of live attenuated vaccines. Fourteen mutants identified by utilizing BLMS showed reduced virulence as compared to wild type E. ictaluri and provided increased protection from ESC compared to non-vaccinated controls. Among the 14 mutants, we observed redundant mutations in two genes. Three different mutants harbored transposon insertion at different locations in gcvP, which encodes glycine cleavage system protein P. Two different mutants harbored transposon insertion at the same location in rseB, which encodes a negative regulator of sigma E activity. This provides confirmation that BLMS procedure is a sensitive method detecting true targets.

BLMS can detect novel virulence relevant genes located on native plasmids or show importance of native plasmids in bacterial virulence if random mutation occurs in the origin of replication of native plasmids. While applying BLMS to E. ictaluri, we observed that at least one of the two native plasmids of E. ictaluri (pEI1) may be important in E. ictaluri virulence because two different locations were targeted on this plasmid in two different mutants (EiAKMut04 and EiAKMut10).

Production of Edwardsiella ictaluri Mutant Library

A library of random transposon insertion mutations in conditionally bioluminescent E. ictaluri strain 93-146 carrying pAKgfplux2 was generated. The library containing approximately 15,000 mutants was created by using a derivative of the mariner transposon Himar1 carried on pMAR2xT7 plasmid. The library consisted of mutants arrayed in 39 384-well plates. A duplicate of the whole library was also prepared. The produced mutant library is compatible with genetic footprinting of the mutants with transposon-site hybridization (TraSH) analysis.

Identification of Serum and Neutrophil Susceptible Edwardsiella ictaluri Mutants

We used the high throughput bioluminescence mutant screening (BLMS) procedure to identify virulence relevant genes of gram negative bacteria in vitro. We screened 2,256 mutants against both serum and neutrophils using BLMS and identified 180 mutants exhibiting light reduction during incubation with these host factors. A second round screening of these 180 mutants in quadruplicate samples allowed us to identify 35 serum, 39 neutrophil, and 26 both serum and neutrophil susceptible mutants for in vivo studies. General outline of the integrated procedures including in vitro BLMS and in vivo fish screening applied can be seen in FIG. 1. Injection of catfish with 100 BLMS selected mutants resulted in identification of 14 attenuated mutants including 8 mutants susceptible to both serum and neutrophils, 4 susceptible to neutrophils, and 2 susceptible to serum, which were further characterized in vivo.

Characterization of Virulence and Vaccine Potentials of Edwardsiella ictaluri Mutants

In vitro BLMS allowed us reduce the number of target mutants to an amenable size for in vivo characterization. Fish were infected with 14 mutants and their attenuation and vaccine potentials were determined (Table 1). Virulence of EiAKMut07 and EiAKMut09 appeared to be higher than other mutants while E. ictaluri wild type was the most virulent in immersion immunization. EiAKMut02, EiAKMut07, EiAKMut10, and EiAKMut12 provided more protection than other mutants. Similarly, EiAKMut07 and EiAKMut09 were also the most virulent strains in the injection immunization though they were attenuated as compared to wild type strain. Virulence of second category of mutants in injection immunization ranged from 1.25% to 11.67%, while the third category of mutants including EiAKMut02, EiAKMut03, EiAKMut04, and EiAKMut06 seemed to be not virulent. After immersion infection, efficacy of EiAKMut02, EiAKMut08, and EiAKMut12 were statistically superior to others.

TABLE 1 Summary of in vivo mutant characterization results Immunization^(Im) Wt challenge^(Im) Immunization^(In) Wt challenge^(Im) Groups % M SE % M SE % M SE % M SE EiAKMut01 — — 1.32 1.32 6.67 3.33 5.46 0.10 EiAKMut02 — — — — — — — — EiAKMut03 — — 6.35 5.01 — — 1.67 1.67 EiAKMut04 — — 2.17 2.17 — — 1.67 1.67 EiAKMut05 — — 1.09 1.09 1.67 1.67 3.33 3.33 EiAKMut06 — — 2.39 1.38 — — 2.63 1.52 EiAKMut07 1.19 1.19 — — 58.33 8.82 3.03 3.03 EiAKMut08 — — 1.32 1.32 1.67 1.67 — — EiAKMut09 1.25 1.25 2.44 1.41 48.33 9.47 4.44 4.44 EiAKMut10 — — — — 11.25 1.26 4.10 2.55 EiAKMut11 — — 22.02 8.63 1.25 1.19 2.72 1.58 EiAKMut12 — — — — 10.00 2.04 — — EiAKMut13 — — 1.00 1.00 11.67 7.25 5.85 0.48 EiAKMut14 — — 18.78 3.80 5.00 0.22 1.75 1.75 EiWt 17.55  8.01 1.39 1.39 83.75 3.75 — — PBS — — 88.73 1.69 — — 12.50  2.50 Im, immersion; In, injection; Wt, wild type E. ictaluri 93-146; M, mortality; SE, standard error; —, no mortality observed.

Identification of MAR2xT7 Insertions in Edwardsiella ictaluri Genome

MAR2XT7 insertion locations were determined using single primer PCR amplification of transposon ends and nested primer sequencing. We determined the insertion locations of MAR2XT7 and identified disrupted genes from all fourteen mutants (Table 2). Gene identification using database searches indicated that glycine cleavage system protein P (gcvP) was disrupted in three of the mutants (EiAKMut02, EiAKMut03, and EiAKMut08) but at different locations. Similarly, negative regulator of sigma E activity (rseB) gene was mutated at the same location in EiAKMut01 and EiAKMut07. Interestingly, two genes located on one of the native plasmids of E. ictaluri (pEI1) were also mutated. One of these genes was a putative RNA one modulator protein while the other was a hypothetical protein.

TABLE 2 Summary of insertion identification results Mutants Type AN Gene ID Location EiAKMut01 NS Negative regulator of sigma E activity (rseB) aaTA{circumflex over ( )}MAR2XT7 EiAKMut02 NS Glycine cleavage system protein P (gcvP) ggTA{circumflex over ( )}MAR2XT7 EiAKMut03 NS Glycine cleavage system protein P (gcvP) taTA{circumflex over ( )}MAR2XT7 EiAKMut04 NS Hypothetical protein pEI1_p1 taTA{circumflex over ( )}MAR2XT7 EiAKMut05 NS Succinate dehydrogenase/fumarate reductase, tcTA{circumflex over ( )}MAR2XT7 cytochrome b subunit EiAKMut06 NS Electron transport complex protein RnfB ggTA{circumflex over ( )}MAR2XT7 EiAKMut07 NS Negative regulator of sigma E activity (rseB) aaTA{circumflex over ( )}MAR2XT7 EiAKMut08 NS Glycine cleavage system protein P (gcvP) acTA{circumflex over ( )}MAR2XT7 EiAKMut09 N Fimbrial chaperon protein ggTA{circumflex over ( )}MAR2XT7 EiAKMut10 N Putative RNA one modulator protein pEI1_p4 atTA{circumflex over ( )}MAR2XT7 EiAKMut11 N 2-oxoglutarate dehydrogenase E1 component agTA{circumflex over ( )}MAR2XT7 EiAKMut12 N Malate dehydrogenase aaTA{circumflex over ( )}MAR2XT7 EiAKMut13 S UDP-glucose 6-dehydrogenase taTA{circumflex over ( )}MAR2XT7 EiAKMut14 S TnpA tcTA{circumflex over ( )}MAR2XT7 NS, neutrophil and serum; N, neutrophil; S, serum; AN, accession number; {circumflex over ( )}, insertion point.

Attenuation and Efficacy of Edwardsiella ictaluri Mutants and AQUAVAC-ESC

We compared our attenuated E. ictaluri mutants with a commercial live attenuated vaccine to determine whether our mutants provide reduced virulence and improved protection against the wild type E. ictaluri infections. As can be seen from FIG. 2 and FIG. 3, attenuation and efficacy experiments indicated that some of our mutants performed better than AQUAVAC-ESC while others did not. Immersion immunization indicated that AQUAVAC-ESC, EiAKMut02, EiAKMut05, EiAKMut08, and EiAKMut13 were completely attenuated while others showed increased attenuation as compared to wild type E. ictaluri. Infection of immunized fish indicated that EiAKMut05 provided the best protection with no mortality in the immunized fish. Six other mutants indicated improved protection as compared to AQUAVAC-ESC. EiAKMut13 performed inferior to commercial vaccine in terms of protection, but EiAKMut13 provided slightly greater protection against wild type infection as compared to the sham vaccinated fish.

EXAMPLE Live Attenuated Vaccine for use in Catfish

The following description more particularly discloses the steps used in practicing the BLMS method as applied to the claimed E. ictaluri live attenuated vaccine.

Bacterial strains, plasmids, and growth conditions. Escherichia coli SM10 λpir was used as the donor strain in conjugations for transfer of pAKgfplux2 and pMAR2XT7 into Edwardsiella ictaluri strain 93-146. E. ictaluri and E. coli DH5α carrying pAKgfplux2 were used as negative and positive controls in neutrophil and serum screening experiments. E. coli strains were grown using Luria-Bertani (LB) broth and agar plates at 37° C. and E. ictaluri was grown using brain heart infusion (BHI) broth and agar plates at 30° C. Antibiotics were added to the following final concentrations: ampicillin (100 μg/ml), colistin (12.5 μg/ml), gentamicin (12.5 μg/ml). 2 mM Isopropyl-β-D-thiogalactopyranosid (IPTG) was used in growth medium and screening assays to induce expression of bacterial luciferase operon (luxCDABE) from the lacZ promoter in pAKgfplux2. E. ictaluri minimal medium was used to eliminate auxotrophic mutants.

Construction of Edwardsiella ictaluri mutant library. MAR2xT7 insertions were generated by introducing pMAR2xT7 from E. coli SM10 λpir into E. ictaluri carrying pAKgfplux2 in conjugal mating as described in Karsi, A. & Lawrence, M. L. Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria. Plasmid 57, 286-295 (2007), herein incorporated by reference. Transposants were selected on 20×20 cm LB agar plates containing 12.5 μg/ml colistin and 12.5 μg/ml gentamicin. Putative transposants were picked robotically using a Flexsys Colony Picker (GENOMIC SOLUTIONS, Ann Arbor, Mich.) into 60 μl of LB broth containing colistin and gentamicin in 384-well microtiter plates and grown overnight in HIGRO shaker (GENOMIC SOLUTIONS) at 30° C. A duplicate library was prepared by the Flexsys Colony Picker before sterile glycerol was robotically added to the cultures at a final concentration of 20%. Plates were sealed with aluminum foil to prevent cross contamination, lids were taped to prevent freeze drying, and libraries were frozen at −80° C.

Catfish serum and neutrophil preparation. Specific pathogen free (SPF) fish facility at the College of Veterinary Medicine, Mississippi State University maintains SPF channel catfish. For serum preparation, 1-2 kg SPF catfish were anesthetized in water containing 200 mg/l tricaine methane sulfonate (MS222) and blood was collected at 1% of body weight. A recovery period of one month was given for subsequent blood collections. Serum was obtained and stored at −80° C. as single use aliquots. Neutrophils were isolated from the single cell suspensions of anterior kidney cells of SPF catfish (38.63±0.68 cm, 424.20±23.34 g) using discontinuous percoll gradient centrifugation procedure described previously. Purity of neutrophils collected from the 1.060-1.080 interface was determined using flow cytometry.

In vitro mutant screening using catfish serum and neutrophils. 384-well plates containing the frozen mutants were taken out from the −80° C. and aluminum cover is removed immediately. Plates were centrifuged briefly and returned to 4° C. until the culture thawed completely. Four 96-well plates containing 195 μl of BHI medium with colistin and gentamicin antibiotics and 2 mM IPTG were prepared. Five microliters of mutant bacteria were inoculated in each well and were grown at 30° C. by shaking at 250 RPM for 16-18 hours. The next day, 10 μl of mutant culture containing approximately 106 CFU was mixed with 90 μl of catfish serum containing 2 mM IPTG and plates were returned to the imaging chamber of an IVIS Imaging System 100 Series (XENOGEN CORP., Alameda, Calif.).

Initial bioluminescence of the serum was detected after five min pre-incubation of samples in the imaging chamber adjusted to 30° C. to eliminate temperature effect on light production. After initial imaging, subsequent images were captured from the same plates at every 15 min intervals during the 90 min data collection. Images were analyzed and photons emitted from each well were quantified using Living Image Software v2.50 (XENOGEN CORP.). Percent light change between the first and last measurement times was determined for each mutant and compared to serum resistant (E. ictaluri 93-146 pAKgfplux2) and serum sensitive (DH5α pAKgfplux2) controls included in each plate.

Neutrophil screening was accomplished by setting up phagocytosis assays including freshly isolated neutrophils with 75% or higher purity, 15% SPF catfish serum, 2 mM IPTG, and mutant bacteria. Neutrophil to bacteria numbers were adjusted to give a ratio between 1:40 and 1:80. Bioluminescence imaging was conducted as described above in the serum screening procedure. Percent bioluminescence change in 2,256 mutants was calculated and compared to those of positive and negative controls.

One hundred and eighty mutants with reduced bioluminescence were re-screened against serum and neutrophils in quadruplicate samples and data were analyzed using General Linear Model procedure of SAS v 9.1 (SAS Institute Inc., Carey N.C.). 100 mutants were selected for in-vivo screening studies.

In vivo mutant screening. SPF channel catfish (5.20±0.18 cm) were transferred from the SPF fish facility to 40 1 flow-through tanks with dechlorinated municipal water. Fish were maintained in well-aerated tanks with a water temperature of 28° C. throughout the experiments. After one weak of acclimation, fish were anesthetized in water containing 100 mg/l MS222 and each mutant was injected into 15 catfish at a concentration of approximately 1×10⁷ CFU in 100 μl phosphate-buffered saline (PBS). Wild-type and PBS injected fish were also included in the experiment as positive and negative controls. Fish were monitored daily and dead fish were removed from the tanks. Percent mortality rates indicated attenuation state of serum, neutrophil, and both serum and neutrophil mutants. Fourteen mutants with the highest attenuation rates were further characterized.

Determination of virulence and vaccine potentials. Virulence and efficacy of 14 mutants were characterized by infecting catfish by both intraperitoneal injection and immersion. Each 40 1 flow-through tank contained twenty fish and four tanks were used for each mutant. Fish were allowed to acclimate for one week. Quadruplicate wild type and PBS controls were also included in all experiments. Bacteria numbers were adjusted to be equal by determining OD₆₀₀ readings and adjusting volumes accordingly. In the first study, fish (13.80±0.26 cm, 25.83±1.49 g) were infected by immersion in water containing 1×10⁶ CFU/ml for one hour. After 21 days, immunized fish were infected with wild-type E. ictaluri by immersion in water with 1×10⁷ CFU/ml for one hour. Fish were monitored and dead fish were removed daily. In the second study, fish (14.61±0.33 cm, 32.70±2.36 g) were anesthetized and infected by injecting 1×10⁵ CFU in 100 μl PBS. After 21 days, fish were infected by immersion as described above. Virulence and efficacy of each mutants and controls were calculated from the fish mortality rates.

Identification of transposon insertion sites. Transposon insertion sites were identified by using a single primer PCR protocol. Mutants were grown for 18 hours and genomic DNA was prepared using WIZARD Genomic DNA Purification Kit (PROMEGA). In the first round of PCR reaction, the transposon specific template was amplified linearly for 40 cycles. A second round produced specific and non-specific amplicons due to low annealing temperature at 30° C. The final round further amplified the amplicons. The 25 μl PCR reaction contained 0.2 mM dNTPs, 0.2 μM transposon specific primer, 1.5 mM MgCl2, buffer, and 1.25 units of Taq polymerase (PROMEGA). The five μl single primer PCR reaction was cleaned with 2 μl of EXOSAP-IT enzyme mix (USB CORP.) according to the manufacturer's instructions. Twenty micoliters of BIGDYE v3.1 sequencing reaction contained 2 μl of EXOSAP-IT enzyme mix treated template and 10 μM nested transposon specific primer. Transposon specific sequences were trimmed and remaining bacterial sequences were searched against nucleotide and protein databases using BLAST program.

Vaccination studies. Virulence and efficacy of mutants were compared to a commercial vaccine (AQUAVAC-ESC). Experiment contained 10 mutants, a mixed group containing Mut02, Mut04, Mut05, and Mut06, a commercial live attenuated vaccine, and wild-type and sham controls. Two of the mutants (Mut02 and Mut08) harbored transposon insertions in the same gene but at different locations and therefore served as an internal control in the experiments. Each 40 1 flow-through tank contained 25 fish and four tanks were assigned to each group. Fish were allowed to acclimate for two weeks before bacterial challenges. Bacteria numbers were adjusted to be equal by determining OD₆₀₀ readings and adjusting volumes accordingly. For vaccination, fish (11.62±0.16 cm, 15.36±0.65 g) were infected by immersion in water containing 2×10⁷ CFU/ml for one hour. After 21 days, immunized fish were infected with wild-type E. ictaluri by immersion in water with 1×10⁷ CFU/ml for one hour. Fish were monitored and dead fish were removed daily. Mean percent mortalities for each group were calculated, arcsine-transformed, and analyzed using PROC GLM procedure of SAS 9.1 (SAS Institute Inc., Carey, N.C.).

Analysis of the Mutants

Succinate-ubiquinone oxidoreductase (SQR) encoded by the sdhCDAB gene cluster and menaquinol-fumarate oxidoreductase (QFR) encoded by the frdABCD gene cluster are part of the trichloroacetic acid (TCA) cycle and are structurally and functionally related membrane-bound enzyme complexes. EiAKMut05 has an insertion in the sdhC gene, which encodes one of four subunits of the succinate dehydrogenase complex. SdhC is one of the two subunits that anchor the complex in the cytoplasmic membrane. Succinate dehydrogenase is part of the aerobic respiratory chain and the Krebs cycle, oxidizing succinate to fumarate while reducing ubiquinone to ubiquinol. It is closely related to fumarate reductase, which catalyzes the reverse reaction. Succinate dehydrogenase and fumarate reductase can replace each other at different relative rates and with different apparent substrate affinities. Because of fumarate reductase's ability to convert succinate to fumarate, sdhCDA mutant of Salmonella enterica serovar Typhimurium were slightly attenuated and complete attenuation was achieved by succinate dehydrogenase/fumarate reductase double mutation. In E. ictaluri, sdhC is the first gene in a polycistronic operon that encodes the four components of succinate dehydrogenase; therefore, it is possible that the mutation in sdhC has a polar effect on expression of downstream genes. Our results indicate that attenuation of E. ictaluri was achieved with sdhC mutation without a need for generating double mutants in frd genes. An explanation for this could be that fumarate reductase's ability to convert succinate to fumarate in E. ictaluri is not as efficient as compared to Salmonella and E. coli or E. ictaluri sdhC mutant is cleared from the fish before bacteria can activate fumarate reductase or anaerobic condition triggering use of fumarate reductase does not occur during fish infection. Our recent analysis of E. ictaluri proteome showed that many proteins involved in Tricarboxylic acid (TCA) pathway including the fumarate reductase complex present and TCA pathway significantly represented in E. ictaluri (unpublished data). In E. coli sdhC mutants, SdhC activity is located in the cytoplasm, and it utilizes artificial electron acceptors; in contrast, wild-type E. coli has membrane-associated SdhC activity with ubiquinone as the electron acceptor. In E. coli, fumarate reductase is expressed under anaerobic conditions with glucose as a carbon source. Although SdhC has similar function, hydrophobicity, and protein size to the membrane-binding subunit from fumarate reductase (FrdC), SdhC and FrdC do not share significant sequence identity. In Helicobacter pylori, fumarate reductase was found to be essential for colonization of mouse gastric mucosa. In E. coli and Salmonella, succinate dehydrogenase is known to contribute to pathogenicity. The organic acids formate and succinate have a protective effect in stationary phase cells against killing effects of antimicrobial peptide BPI, which appears to disrupt the bacterial respiratory chain. Maintenance of protective levels of formate and succinate requires the activity of formate dehydrogenase and succinate dehydrogenase, respectively. E. ictaluri also encodes the formate dehydrogenase complex in its genome.

Mutants 2, 3, and 8 all had insertions in gcvP, which encodes a protein that is part of the glycine cleavage system. The glycine cleavage system is a loosely associated four subunit enzyme complex that catalyzes the reversible oxidation of glycine to form 5,10-methylenetetrahydrofolate, which serves as a one carbon donor. It is one of two sources of 1 C units with serine hydroxymethyltransferase being the other (and is considered the more important source). Expression of the glycine cleavage enzyme system is induced by glycine, and gcv mutants are unable to use glycine as a 1 C source and excrete glycine. The glycine cleavage system is also part of the formyltetrahydrofolate biosynthesis system. GcvP is a 104 kDa protein that catalyzes the decarboxylation of glycine. In E. ictaluri, gcvP is the third gene in a three gene operon; it is located downstream of gcvH and gcvT, which encode subunits of the glycine cleavage system. E. ictaluri also has a gene that encodes serine hydroxymethyltransferase. The glycine cleavage system has not been linked with virulence previously, and our disclosed composition and method are the first to employ it.

Mutant 1 had an insertion in rseB, which encodes one of two negative regulators of sigmaE. RseA is considered the major regulator of sigmaE. SigmaE is expressed in response to heat shock and other stresses on membrane and periplasmic proteins, including misfolding of outer membrane proteins, hyperosmotic stress, metal ion exposure, changes in LPS structure, and starvation signal ppGpp. SigmaE is required for heat-induced transcription of rpoH, which encodes heat shock factor sigma32 and other heat shock proteins. RseB is a periplasmic protein that interacts with RseA. RseB stimulates binding of RseA to sigmaE, thereby assisting RseA in tethering sigmaE to the cytoplasmic membrane. Degradation of RseA releases sigmaE and allows it to interact with the core enzyme of RNA polymerase to initiate transcription. Although mutations in rseA cause increased sigmaE activity, an rseB mutant shows wild-type sigmaE activity under inducing conditions and exhibits a small increase in sigmaE activity under non-inducing conditions. In E. ictaluri, rseB is the third gene in a polycistronic operon. It is downstream of rpoE, which encodes sigmaE, and rseA, and it is upstream of rseC, which encodes a positive regulator of sigmaE. SigmaE is required for Salmonella virulence and mediates Salmonella resistance to oxidative stress and antimicrobial peptides. SigmaE is also required for Salmonella to survive intracellularly. We disclose the first report of RseB being associated with virulence.

Mutant 6 has an insertion in rsxB, which encodes one of six proteins that form a SoxR reducing system in E. coli. SoxR is a regulatory protein that senses superoxide and nitric oxide and induces expression of an oxidative stress response. When SoxR is activated by oxidation of its [2Fe-2S] cluster, it induces expression of SoxS, which is a transcriptional regulator that induces expression of superoxide dismutase and other oxidative response proteins. The SoxR reducing system inactivates SoxR, thereby turning off the oxidative stress response. In E. coli, when any of the six rsx genes are mutated, SoxS is constitutively expressed, leading to induction of oxidative stress response. In Salmonella, SoxS is not essential for virulence, but SoxS was found to contribute to virulence in an E. coli mouse pyelonephritis model. In E. ictaluri, rsxB is the second in the six gene rsx operon.

Mutant 4 has an insertion in a gene encoding a hypothetical protein located on one of the two E. ictaluri constitutive plasmids, pEI1. The protein has >50% identity with Salmonella effector proteins with leucine rich repeats that are secreted through a type III secretion system. The 618 amino acid protein appears to be in a monocistronic operon.

EXAMPLE Identifying Mutants that Fail to Attach to the Host Epithelium

The BLMS method can also be used to identify bacterial mutants that fail to attach to the host molecules, cells, or surfaces. Attachment and colonization of the host epithelium is an indispensable first step to any bacterial infection and can be achieved through a variety of diverse mechanisms. To investigate these attachment mechanisms in Edwardsiella ictaluri, we used random insertion of the pMar2xT7 transposon to generate a library of 1728 mutants. Each mutant expressed bioluminescence constitutively from the plasmid pAKlux1. This library was then screened in a high throughput fashion using an IVIS Living Image System (XENOGEN) in a series of nested in vivo challenges using a skin abrasion model we developed. Twenty mutants that displayed a decrease in their ability to colonize the channel catfish epithelium were identified. Results from this study will delineate mechanisms of E. ictaluri attachment to channel catfish skin and could lead to improved methods for prevention of enteric septicemia of catfish.

Combinations of mutations. Combinations of mutations can be constructed using the pathways we have disclosed. Specifically, in-frame deletions in TCA cycle enzymes and glycine cleavage system protein can be constructed to create greater attenuation while retaining antigenicity. Mutation of the glycine cleavage system as a vaccine strategy is a new strategy that has never been previously reported. Thus, our patenting potential is very strong for vaccine development based on glycine cleavage system. EiAKMut2 has a mutation in gcvP. The glycine cleavage system functions in providing 5,10-methylenetetrahydrofolate as a source for 1 C moieties. Our plan is to construct a mutant containing deletions in gcvP (our current mutant) and in another enzyme that serves to provide 5,10-methylenetetrahydrofolate through an alternative pathway. Knocking out both pathways should cause improved attenuation.

Mutation of genes encoding TCA cycle enzymes, exemplified by EiAKMut5 and EiAKMut12, shows great potential as a strategy for an effective live attenuated E. ictaluri vaccine. Knockout of genes encoding TCA cycle enzymes was recently discovered as an effective strategy for vaccine development in Salmonella (which is closely related to Edwardsiella). We have found that knocking out a single TCA cycle gene does not always cause complete attenuation, but knocking out two genes can cause complete attenuation. Specifically, a Salmonella sdhCDA-frdABCD mutant was fully avirulent and effective as a vaccine, while a Salmonella sdhCDA mutant was not fully attenuated. A combination mutant can be constructed that has deletions in sdhC (the gene mutated in EiAKMut5) and mdh (the gene mutated in EiAKMut12), as well as a second sdhC combination mutant that has a knockout in another enzyme that encodes a related TCA cycle enzyme.

Use of Other Bacterial Species as Live Attenuated Vaccines for Various Hosts

The method and compositions disclosed herein are not limited to Edwardsiella ictaluri, but can be used in other bacteria as well. Because the genes discovered in this research project are well conserved in bacteria, the mutation of these genes in other bacterial pathogens can be utilized for development of effective live attenuated vaccines to prevent other diseases. For example, Salmonella enterica is closely related to Edwardsiella ictaluri and is in the same bacterial family (Enterobacteriaceae). The pathogenesis of salmonellosis in mammals is also similar to the pathogenesis of enteric septicemia of catfish caused by E. ictaluri. The mutation of these genes in Salmonella will result in development of an effective live attenuated vaccine for prevention of salmonellosis in various animal hosts. Similarly, the genus Yersinia is also in the same family as Edwardsiella and Salmonella, and the disease pathogenesis of Yersinia is similar to enteric septicemia of catfish. Therefore, the mutation of these genes will be effective for development of live attenuated vaccines for Yersinia pestis, which causes bubonic plague in humans, Y. enterocolitica and Y. pseudotuberculosis, which cause gastrointestinal disease in humans and other mammals, and Y. ruckeri, which causes enteric redmouth disease in salmonid fish.

The mutation of these genes may be an effective strategy for development of live attenuated vaccines for pathogenic Escherichia coli, Shigella flexneri, and Shigella dysenterieae, which are also closely related to E. ictaluri. The mutation of these genes can also be used for development of live attenuated vaccines against Francisella tularensis, which causes tularemia in humans, because the disease pathogenesis is similar to enteric septicemia of catfish. Other bacterial pathogens that we anticipate mutation of these genes may be effective for development of live attenuated vaccines include Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Haemophilus influenzae, Haemophilus ducreyi, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Actinobacillus actinomycetemcomitans, Avibacterium paragallinarum, Moraxella catarrhalis, Moraxella bovis, Pseudomonas aeruginosa, Coxiella burnetii, Bordetella bronchiseptica, Bordetella pertussis, Bordetella parapertussis, Bordetella avium, Burkholderia mallei, Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Brucella abortus, Legionella pneumophila, Helicobacteri pylori, and Campylobacter jejuni. Mutation of these genes may also be effective for development of live attenuated vaccines for gram-positive pathogens such as Listeria monocytogenes. In addition, BLMS may be an effective tool for identification of new gene targets for development of live attenuated vaccines.

Although the present invention has been described and illustrated with respect to at least one preferred embodiment and uses therefore, it is not to be so limited since modifications and changes can be made therein which are within the full intended scope of the invention. 

We claim:
 1. A composition for providing immunological protection from diseases caused by Edwardsiella ictaluri which comprises a live attenuated strain of bacteria, wherein said live attenuated strain of bacteria is selected by introducing random transposon mutations into bacterial strains expressing bacterial luciferase operon and screening for said mutations by measuring reduction in bioluminescence.
 2. A composition for providing immunological protection from diseases caused by Edwardsiella ictaluri which comprises a live attenuated strain of the bacteria selected from the list of mutations consisting of: EiAKMut01, EiAKMut02, EiAKMut03, EiAKMut04, EiAKMut05, EiAKMut06, EiAKMut07, EiAKMut08, EiAKMut09, EiAKMut10, EiAKMut11, EiAKMut12, EiAKMut13, and EiAKMut14.
 3. A live attenuated vaccine prepared comprising a composition created by introducing random transposon mutations into bacterial strains expressing bacterial luciferase operon and screening for said mutations by measuring reduction in bioluminescence.
 4. A composition for providing immunological protection from diseases caused by Edwardsiella ictaluri which comprises a live attenuated strain of the bacteria having at least one mutation in the genes coding for glycine cleavage system (gcvP), serine hydroxymethyltransferase, succinate dehydrogenase, malate dehyrogenase, 2-oxoglutarate dehydrogenase, negative regulator of sigma E activity (rseB), hypothetical protein pEI1_p1, electron transport complex protein RnfB, Fimbrial chaperon protein, Putative RNA one modulator protein pEI1_p4, or UDP-glucose 6-dehyrogenase, fumarate reductase (frdA), and genes encoding enzymes in the tricarboxylic acid (TCA) cycle.
 5. A composition for providing immunological protection from diseases caused by bacteria which comprises a live attenuated strain of the bacteria having at least one mutation in the genes coding for glycine cleavage system (gcvP), serine hydroxymethyltransferase, succinate dehydrogenase, malate dehyrogenase, 2-oxoglutarate dehydrogenase, negative regulator of sigma E activity (rseB), hypothetical protein pEI1_p1, electron transport complex protein RnfB, Fimbrial chaperon protein, Putative RNA one modulator protein pEI1_p4, or UDP-glucose 6-dehyrogenase, fumarate reductase (frdA), and genes encoding enzymes in the tricarboxylic acid (TCA) cycle.
 6. The composition of claim 5 wherein the bacteria is of the genus Yersinia.
 7. The composition of claim 5 wherein the bacteria is of the genus Salmonella.
 8. The composition of claim 5 wherein the bacteria is of the genus Shigella.
 9. The composition of claim 5 wherein the bacteria is Escherichia coli.
 10. The composition of claim 5 wherein the bacteria is selected from the list consisting of Francisella tularensis, Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Haemophilus influenzae, Haemophilus ducreyi, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Actinobacillus actinomycetemcomitans, Avibacterium paragallinarum, Moraxella catarrhalis, Moraxella bovis, Pseudomonas aeruginosa, Coxiella burnetii, Bordetella bronchiseptica, Bordetella pertussis, Bordetella parapertussis, Bordetella avium, Burkholderia mallei, Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Brucella abortus, Legionella pneumophila, Helicobacteri pylori, Campylobacter jejuni, and Listeria monocytogenes. 